A direct solubilization procedure for the determination of DNA and protein in cultured fibroblast monolayers

A procedure for the direct solubilization of fibroblast monolayers allowing precise determination of DNA and protein content has been developed. This simple procedure is applicable to the analysis of these macromolecules in fibroblast cultures containing small numbers of cells (<1 × 10⁵).     

The regulation of the decay of nitrate reductase. Evidence for the existence of at least two mechanisms of decay.

There seem to be at least two different mechanisms of decay of nitrate reductase in Neurospora in vivo: one which is very sensitive to EDTA and cycloheximide, decreases with mycelial age and is not increased by an increase in temperature from 27 to 37 degrees C, the other which is relatively insensitive to EDTA and cycloheximide, increases with the age of the mycelium and with the above temperature shift.     

Pyrrolovesamicols--synthesis, structure and VAChT binding of two 4-fluorobenzoyl regioisomers

This Letter describes the synthesis of two regioisomers of a new class of vesamicol analogs as possible ligands for imaging the vesicular acetylcholine transporter in future PET studies. The two pyrrolovesamicols (±)-6a and (±)-6b were synthesized by nucleophilic ring opening reaction of a tetrahydroindole epoxide precursor with 4-phenylpiperidine. The reaction mechanism of the synthesis was studied by HPLC and the molecular structures were determined by X-ray structure analysis. Unexpected low binding affinities to VAChT (K(i)=312±73 nM for (±)-6a and K(i)=7320±1840 nM for (±)-6b) were determined by competitive binding analysis using a cell line stably transfected with ratVAChT and (-)-[(3)H]vesamicol.     

Fuzzy Logic Analysis of Kinase Pathway Crosstalk in TNF/EGF/Insulin-Induced Signaling

When modeling cell signaling networks, a balance must be struck between mechanistic detail and ease of interpretation. In this paper we apply a fuzzy logic framework to the analysis of a large, systematic dataset describing the dynamics of cell signaling downstream of TNF, EGF, and insulin receptors in human colon carcinoma cells. Simulations based on fuzzy logic recapitulate most features of the data and generate several predictions involving pathway crosstalk and regulation. We uncover a relationship between MK2 and ERK pathways that might account for the previously identified pro-survival influence of MK2. We also find unexpected inhibition of IKK following EGF treatment, possibly due to down-regulation of autocrine signaling. More generally, fuzzy logic models are flexible, able to incorporate qualitative and noisy data, and powerful enough to produce quantitative predictions and new biological insights about the operation of signaling networks.     

Towards a Rigorous Assessment of Systems Biology Models: The DREAM3 Challenges

Background

Systems biology has embraced computational modeling in response to the quantitative nature and increasing scale of contemporary data sets. The onslaught of data is accelerating as molecular profiling technology evolves. The Dialogue for Reverse Engineering Assessments and Methods (DREAM) is a community effort to catalyze discussion about the design, application, and assessment of systems biology models through annual reverse-engineering challenges.

Methodology and Principal Findings

We describe our assessments of the four challenges associated with the third DREAM conference which came to be known as the DREAM3 challenges: signaling cascade identification, signaling response prediction, gene expression prediction, and the DREAM3 in silico network challenge. The challenges, based on anonymized data sets, tested participants in network inference and prediction of measurements. Forty teams submitted 413 predicted networks and measurement test sets. Overall, a handful of best-performer teams were identified, while a majority of teams made predictions that were equivalent to random. Counterintuitively, combining the predictions of multiple teams (including the weaker teams) can in some cases improve predictive power beyond that of any single method.

Conclusions

DREAM provides valuable feedback to practitioners of systems biology modeling. Lessons learned from the predictions of the community provide much-needed context for interpreting claims of efficacy of algorithms described in the scientific literature.     

Collecting and organizing systematic sets of protein data

Systems biology, particularly of mammalian cells, is data starved. However, technologies are now in place to obtain rich data, in a form suitable for model construction and validation, that describes the activities, states and locations of cell-signalling molecules. The key is to use several measurement technologies simultaneously and, recognizing each of their limits, to assemble a self-consistent compendium of systematic data.     

Repression of nitrate reductase in Neurospora studied by using L-methionine-DL-sulfoximine and glutamine auxotroph gln-1b.

The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-1b, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.     

Binding of the essential Saccharomyces cerevisiae kinetochore protein Ndc10p to CDEII

Chromosome segregation at mitosis depends critically on the accurate assembly of kinetochores and their stable attachment to microtubules. Analysis of Saccharomyces cerevisiae kinetochores has shown that they are complex structures containing >/=50 protein components. Many of these yeast proteins have orthologs in animal cells, suggesting that key aspects of kinetochore structure have been conserved through evolution, despite the remarkable differences between the 125-base pair centromeres of budding yeast and the Mb centromeres of animal cells. We describe here an analysis of S. cerevisiae Ndc10p, one of the four protein components of the CBF3 complex. CBF3 binds to the CDEIII element of centromeric DNA and initiates kinetochore assembly. Whereas CDEIII binding by Ndc10p requires the other components of CBF3, Ndc10p can bind on its own to CDEII, a region of centromeric DNA with no known binding partners. Ndc10p-CDEII binding involves a dispersed set of sequence-selective and -nonselective contacts over approximately 80 base pairs of DNA, suggesting formation of a multimeric structure. CDEII-like sites, active in Ndc10p binding, are also present along chromosome arms. We propose that a polymeric Ndc10p complex formed on CDEII and CDEIII DNA is the foundation for recruiting microtubule attachment proteins to kinetochores. A similar type of polymeric structure on chromosome arms may mediate other chromosome-spindle interactions.